Cotton transgenic event mon 88701 and methods of use thereof

ABSTRACT

The invention provides cotton event MON 88701, and plants, plant cells, seeds, plant parts, and commodity products comprising event MON 88701. The invention also provides polynucleotides specific for event MON 88701 and plants, plant cells, seeds, plant parts, and commodity products comprising polynucleotides specific for event MON 88701. The invention also provides methods related to event MON 88701.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/469,118, filed on Mar. 30, 2011, herein incorporated by reference inits entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “MONS302US.txt”, whichis 21 kilobytes (size as measured in Microsoft Windows®) and was createdon Mar. 9, 2012, is filed herewith by electronic submission and isincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to transgenic Gossypium hirsutum event MON 88701.The event exhibits tolerance to dicamba and glufosinate herbicides. Theinvention also relates to plants, plant parts, plant seeds, plant cells,agricultural products, and methods related to event MON 88701 andprovides nucleotide molecules that are unique to the event and werecreated in connection with the insertion of transgenic DNA into thegenome of a Gossypium hirsutum plant.

BACKGROUND OF THE INVENTION

Cotton (Gossypium hirsutum) is an important crop in many areas of theworld, and the methods of biotechnology have been applied to this cropin order to produce cotton with desirable traits. One such desirabletrait is herbicide tolerance. The expression of an herbicide tolerancetransgene in a plant can confer the desirable trait of herbicidetolerance on the plant, but expression of the transgene may beinfluenced by the chromosomal location and the genomic result of thetransgene insertion. For example, it has been observed in plants thatthere often is variation in the level and pattern of transgeneexpression among individual events that differ in the chromosomalinsertion site of the transgene but are otherwise identical. There mayalso be undesirable and/or desirable phenotypic or agronomic differencesbetween events. Because of this, it is often necessary to produce andanalyze a large number of individual plant transformation events inorder to select an event having both the desirable trait and the optimalphenotypic and agricultural characteristics necessary to make itsuitable for commercial purposes. Such selection often requiresgreenhouse and field trials with many events over multiple years, inmultiple locations, and under a variety of conditions so that asignificant amount of agronomic, phenotypic, and molecular data may becollected. The resulting data and observations must then be analyzed byteams of scientists and agronomists with the goal of selecting acommercially suitable event. Such an event, once selected, may then beused for introgres sing the desirable trait into other geneticbackgrounds using plant breeding methods, and thus producing a number ofdifferent crop varieties that contain the desirable trait and aresuitably adapted to specific local growing conditions.

SUMMARY OF THE INVENTION

The invention provides transgenic cotton plants comprising event MON88701, which exhibit commercially acceptable tolerance to applicationsof dicamba and glufosinate herbicides, having a representative seedsample deposited with the American Type Culture Collection (ATCC®) underPatent Deposit Designation PTA-11754. The invention also provides novelDNA molecules related to cotton event MON 88701 and methods of usingthese molecules. The invention also provides seeds, progeny, plantparts, cells, and commodity products of cotton plants comprising eventMON 88701. The invention also provides methods of using cotton event MON88701 and methods of producing cotton tolerant to both dicamba andglufosinate herbicides.

The invention provides recombinant DNA molecules related to cotton eventMON 88701. These recombinant DNA molecules may comprise nucleotidemolecules having a nucleotide sequence representing a region of thegenomic DNA flanking the transgene insertion, and/or a region of thetransgene insertion, and/or a contiguous sequence of any of theseregions such as a region of the junction between the transgene insertionand flanking genomic DNA of cotton event MON 88701. The invention alsoprovides DNA molecules useful as primers and probes diagnostic forcotton event MON 88701 and amplicons diagnostic for the presence ofcotton event MON 88701. Cotton plants, plant cells, plant parts,commodity products, progeny, and seeds comprising these molecules arealso disclosed.

The invention provides methods, compositions, and kits useful fordetecting the presence and/or absence of DNA derived from cotton eventMON 88701 and thus the presence and/or absence of the event. Theinvention provides a method for detection of MON 88701 by contacting asample comprising DNA with a primer set that when used in a nucleic acidamplification reaction with genomic DNA from a cotton plant or seedcomprising event MON 88701 produces an amplified DNA diagnostic forcotton event MON 88701, performing a nucleic acid amplification reactionthereby producing the amplified DNA, and detecting the presence and/orabsence of the amplified DNA. The invention also provides a method fordetection of MON 88701 by contacting a sample comprising DNA with aprobe that when used in a hybridization reaction with DNA from cottonevent MON 88701 hybridizes to a DNA molecule specific for cotton eventMON 88701, performing a hybridization reaction, and detecting thehybridization of the probe to the DNA molecule. Kits comprising themethods and compositions of the invention useful for detecting thepresence of DNA derived from cotton event MON 88701 are also provided.

The invention provides a cotton plant, seed, plant cell, progeny plant,plant part, or commodity product derived from a plant, plant cell, orseed comprising cotton event MON 88701. The invention also provides acotton plant, seed, plant cell, progeny plant, plant part, or commodityproduct comprising a recombinant DNA molecule having a nucleotidesequence selected from the group consisting of SEQ ID NO: 1-10, andcomplements and fragments thereof. The invention also provides a cottonplant, seed, plant cell, progeny plant, plant part, or commodity productderived from the plant or seed comprising cotton event MON 88701 andcomprising a recombinant DNA molecule that produces an amplified DNAmolecule comprising a sequence selected from SEQ ID NO: 1-10 in a DNAamplification method.

The invention provides a method for controlling weeds in a field byplanting cotton plants comprising event MON 88701 and then applying aneffective dose of dicamba, glufosinate, or both dicamba and glufosinateherbicides capable of controlling the weeds without injuring the cottonevent MON 88701 containing plants. The invention also provides a methodfor controlling weeds in a field by applying an effective dose of atleast dicamba, glufosinate, or dicamba and glufosinate herbicides tocontrol weeds in a field and then planting cotton plants comprisingevent MON 88701 in the field. The invention also provides a method forproducing cotton seed or lint essentially free of weed seeds by plantingseeds of a dicamba and glufosinate tolerant cotton plants comprising MON88701 in a field, applying to the field a post-emergence effective doseof at least dicamba, glufosinate, or dicamba and glufosinate herbicidessufficient to kill the weed species, and harvesting seed or lint fromthe field.

The invention provides methods of producing a cotton plant and/or seedthat tolerates application of dicamba and glufosinate herbicides bysexually crossing a cotton event MON 88701 containing plant comprising asequence selected from SEQ ID NO: 1-10 with a second cotton plant,thereby producing seed, growing the seed to produce progeny plants,treating the progeny plants with dicamba and/or glufosinate, andselecting a progeny plant that is tolerant to both dicamba andglufosinate. The methods may also include selfing the selected progenyplant to produce a plurality of second generation progeny plants andselecting from these a dicamba and glufosinate tolerant plant. Themethods may also include sexually crossing the selected progeny plantwith another cotton plant to produce seed, growing the seed to produce asecond generation of progeny plants, treating the second generation ofprogeny plants with dicamba and/or glufosinate, and selecting a secondgeneration progeny plant that is tolerant to dicamba and glufosinate.The invention provides methods of producing a cotton plant and/or seedthat tolerates application of dicamba and glufosinate herbicides byselfing a dicamba and glufosinate tolerant cotton plant comprising eventMON 88701 comprising a sequence selected from SEQ ID NO: 1-10, therebyproducing seed, growing the seed to produce progeny plants, treating theprogeny plants with dicamba and/or glufosinate; and selecting a progenyplant that is tolerant to dicamba and glufosinate. The inventionprovides methods of determining the zygosity of a cotton event MON 88701containing plant or seed comprising contacting a cotton DNA sample witha primer set comprising SEQ ID NO: 11, 12, and 14 and a probe setcomprising SEQ ID NO: 13 and 15; then performing a nucleic acidamplification reaction with the sample, primer set, and probe set; thendetecting in the nucleic acid amplification reaction a first fluorescentsignal that is diagnostic for event MON 88701 and a second fluorescentsignal different from the first fluorescent signal and that isdiagnostic for native cotton genomic DNA corresponding to the locationof insertion of the event MON 88701 transgene; and analyzing thepresence and/or absence of the first fluorescent signal and the secondfluorescent signal in the nucleic acid amplification reaction, whereinthe presence of both fluorescent signals indicates the sample isheterozygous for event MON 88701 and the presence of only the firstfluorescent signal indicates the sample is homozygous for event MON88701. The invention also provides a cotton plant, seed, plant cell, orplant part comprising dicamba and glufosinate tolerance genes mapped onchromosome A08 at the map position of 19.3 cM and bordered by NG0207927at the map position of 18.6 cM on left and by NG0207529 at the mapposition of 20.0 cM on right, and methods of using the same. Theforegoing and other aspects of the invention will become more apparentfrom the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the organization of the transgenic insert in thegenome of a cotton plant comprising event MON 88701. [A] corresponds tothe relative position of SEQ ID NO: 1; [A′] corresponds to the relativeposition of SEQ ID NO: 3; [A″] corresponds to the relative position ofSEQ ID NO: 5; [B] corresponds to the relative position of SEQ ID NO: 2;[B′] corresponds to the relative position of SEQ ID NO: 4; [B″]corresponds to the relative position of SEQ ID NO: 6; [C] corresponds tothe relative position of SEQ ID NO: 7; [D] corresponds to the relativeposition of SEQ ID NO: 8; [E] corresponds to the relative position ofSEQ ID NO: 9; [F] corresponds to the relative position of SEQ ID NO: 10;SQ21654 and SQ23205 correspond to the relative position of primers usedto identify cotton event MON 88701.

FIG. 2 shows two years of agronomic yield field trial data reported aspounds of seedcotton per acre.

FIG. 3 shows first year yield field trial data reported as pounds ofseedcotton per acre.

FIG. 4 shows second year yield field trial data reported as pounds ofseedcotton per acre with increasing amounts of either dicamba (4.A.) orglufosinate (4.B.) herbicide.

FIG. 5 shows percentage of necrotic spotting occurring with increasingamounts of either dicamba (5.A.) or glufosinate (5.B.) herbicide.

FIG. 6 shows yield field trial data as pounds of seedcotton per acre ofthe spray regimen for event MON 88701.

FIG. 7 shows plants comprising event MON 88701 versus nontransgenicCoker 130 plants sprayed with dicamba and glufosinate in the field.Herbicide applications were 1 lb dicamba (2×) at pre; 1 lb glufosinate(2×) at 2-leaf; 1 lb dicamba at 5-leaf; and 1 lb glufosinate at 8 leaf.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a twenty nucleotide sequence representing the 5′junction region of cotton genomic DNA and the integrated transgenicexpression cassette. SEQ ID NO: 1 is positioned in SEQ ID NO: 10 atnucleotide position 1117-1136.

SEQ ID NO: 2 is a twenty nucleotide sequence representing the 3′junction region of cotton genomic DNA and the integrated transgenicexpression cassette. SEQ ID NO: 2 is positioned in SEQ ID NO: 10 atnucleotide position 5222-5241.

SEQ ID NO: 3 is a sixty nucleotide sequence representing the 5′ junctionregion of cotton genomic DNA and the integrated transgenic expressioncassette.

SEQ ID NO: 4 is a sixty nucleotide sequence representing the 3′ junctionregion of cotton genomic DNA and the integrated transgenic expressioncassette.

SEQ ID NO: 5 is a one-hundred nucleotide sequence representing the 5′junction region of cotton genomic DNA and the integrated transgenicexpression cassette.

SEQ ID NO: 6 is a one-hundred nucleotide sequence representing the 3′junction region of cotton genomic DNA and the integrated transgenicexpression cassette.

SEQ ID NO: 7 is the 5′ sequence flanking the inserted DNA of MON 88701up to and including a portion of integrated transgenic expressioncassette.

SEQ ID NO: 8 is the 3′ sequence flanking the inserted DNA of MON 88701up to and including a portion of integrated transgenic expressioncassette.

SEQ ID NO: 9 is the sequence of the integrated transgenic expressioncassette.

SEQ ID NO: 10 is the contiguous nucleotide sequence of the 5′ sequenceflanking the inserted DNA (SEQ ID NO: 7), the integrated transgenicexpression cassette (SEQ ID NO: 9), and the 3′ sequence flanking theinserted DNA (SEQ ID NO: 8). SEQ ID NO: 10 includes SEQ ID NO: 1-9.

SEQ ID NO: 11 is the sequence of a primer referred to as Primer SQ21654and used to identify cotton event MON 88701. It is complimentary to theinserted expression cassette at the region close to the 3′ transgeneinsertion border. A PCR amplicon produced from a TAQMAN® (PE AppliedBiosystems, Foster City, Calif.) assay using the combination of primersSQ21654 and SQ23205 (SEQ ID NO: 12) is a positive result for thepresence of the event MON 88701. This primer set may also be used toidentify a MON 88701 event in a zygosity assay.

SEQ ID NO: 12 is the sequence of a primer referred to as Primer SQ23205and used to identify cotton event MON 88701. It is complimentary to a 3′region flanking the inserted expression cassette and close to thetransgene DNA insertion border. A PCR amplicon produced from a TAQMAN®(PE Applied Biosystems, Foster City, Calif.) assay using the combinationof primers SQ21654 (SEQ ID NO: 11) and SQ23205 is a positive result forthe presence of the event MON 88701. This is also the primer used toidentify MON 88701 event and wild-type with a zygosity assay.

SEQ ID NO: 13 is the sequence of a probe referred to as Probe PB10280and used to identify cotton event MON 88701. It is complimentary to aregion of the inserted expression cassette and adjacent to the 3′junction of the genomic DNA. This probe is a 6-FAM™-labeled syntheticoligonucleotide. Release of a fluorescent signal in an amplificationreaction using primers SQ21654 and SQ23205 (SEQ ID NO: 11-12) incombination with 6-FAM™-labeled probe PB10280 is diagnostic of event MON88701 in a TAQMAN® assay. PB10280 is also the probe used to identify aMON 88701 event in a zygosity assay.

SEQ ID NO: 14 is the sequence of a primer referred to as Primer SQ23901and used to identify a cotton wild-type allele in a MON 88701 zygosityassay.

SEQ ID NO: 15 is the sequence of a VIC™-labeled probe referred to asProbe PB10631 and used to identify a cotton wild-type allele in a MON88701 zygosity assay.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define theinvention and to guide those of ordinary skill in the art in thepractice of the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art.

The invention provides a transgenic cotton event MON 88701 that exhibitscommercially acceptable tolerance to applications of dicamba andglufosinate herbicides. The event comprises a single insertion oftransgenic DNA into the chromosome/genome of the cotton germplasm. An“event” is produced by: (i) transformation of a plant cell with anucleic acid construct that includes a transgene of interest, (ii)regeneration of a population of plants resulting from the insertion ofthe transgene into the genome of the plant, and (iii) selection of aparticular plant characterized by insertion of the transgene into aparticular location in the plant's genome.

The term “event” refers to a DNA molecule comprising the inserted DNAand the flanking cotton genomic DNA immediately adjacent to either sideof the inserted DNA. This DNA molecule is created by the act ofinserting the transgenic DNA into the genome of the cotton plant, i.e.,by the act of transformation. This DNA molecule therefore comprises anucleotide sequence that is both specific to the event and that isunique to the genome of the cotton plant into which the transgenic DNAhas been inserted, in that this nucleotide sequence contains both thesequence of a particular region of cotton genomic DNA and of thetransgenic DNA insert. The arrangement of the inserted DNA in cottonevent MON 88701 in relation to the surrounding cotton plant genome DNAis therefore specific and unique for cotton event MON 88701. This DNAmolecule is also an integral part of the cotton chromosome of event MON88701 containing plants and as such is static in the plant and may bepassed on to progeny of the plant.

The present invention also provides the original transformant thatincludes the transgene inserted into the particular location in theplant's genome and progeny of the transformant that include thetransgene inserted into the particular location in the plant's genome.Such progeny may be produced by a sexual outcross between thetransformant, or its progeny, and another plant. Such other plant may bea transgenic plant comprising the same or different transgene and/or anontransgenic plant, such as one from a different variety. Even afterrepeated back-crossing to a recurrent parent, the inserted DNA andflanking DNA from the transformed parent is present in the progeny ofthe cross at the same genomic location.

As used herein, the term “cotton” means Gossypium hirsutum and includesall plant varieties that can be bred with cotton, including wild cottonspecies as well as those plants belonging to Gossypium that permitbreeding between species.

Event MON 88701 comprises an integrated transgenic expression cassettethat confers tolerance to applications of dicamba and glufosinateherbicides to the cotton plant. “Dicamba” refers to3,6-dichloro-2-methoxybenzoic acid. Dicamba is a synthetic auxinherbicide useful for controlling broadleaf weeds. Cotton plants weretransformed with a gene (dmo) from Stenotrophomonas maltophilia encodingdicamba mono-oxygenase (DMO). DMO is an enzyme that catalyzes thedeactivation of dicamba via an O-demethylation reaction to thenonherbicidal compound 3,5-dichlorosalicylic acid. “Glufosinate” refersto 2-amino-4-(hydroxymethylphosphinyl)butanoic acid. Glufosinate is anorganophosporus herbicide useful for controlling a broad spectrum ofannual and perennial grass and broadleaf weeds. Cotton plants weretransformed with a Bialaphos Resistance gene (bar) from Streptomyceshygroscopicus encoding phosphinothricin acetyl transferase (PAT). PAT isan enzyme that catalyzes acetylation and thus inactivation ofglufosinate.

As used herein, the term “recombinant” refers to a form of DNA and/orprotein and/or an organism that would not normally be found in natureand as such was created by human intervention. Such human interventionmay produce a recombinant DNA molecule and/or a recombinant plant. Asused herein, a “recombinant DNA molecule” is a DNA molecule comprising acombination of DNA molecules that would not naturally occur together andis the result of human intervention, e.g., a DNA molecule that iscomprised of a combination of at least two DNA molecules heterologous toeach other, and/or a DNA molecule that is artificially synthesized andcomprises a polynucleotide sequence that deviates from thepolynucleotide sequence that would normally exist in nature, and/or aDNA molecule that comprises a transgene artificially incorporated into ahost cell's genomic DNA and the associated flanking DNA of the hostcell's genome. An example of a recombinant DNA molecule is a DNAmolecule described herein resulting from the insertion of the transgeneinto the cotton genomic DNA, which may ultimately result in theexpression of a recombinant RNA and/or protein molecule in thatorganism. As used herein, a “recombinant plant” is a plant that wouldnot normally exist in nature, is the result of human intervention, andcontains a transgene and/or heterologous DNA molecule incorporated intoits genome. As a result of such genomic alteration, the recombinantplant is distinctly different from the related wildtype plant. Anexample of a recombinant plant is a cotton plant described herein ascomprising event MON 88701.

As used herein, the term “transgene” refers to a nucleotide moleculeartificially incorporated into a host cell's genome. Such transgene maybe heterologous to the host cell. The term “transgenic plant” refers toa plant comprising such a transgene.

As used herein, the term “heterologous” refers to a first molecule notnormally found in combination with a second molecule in nature. Forexample, a molecule may be derived from a first species and insertedinto the genome of a second species. The molecule would thus beheterologous to the host and artificially incorporated into a hostcell's genome.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither first nor second DNA molecule would normally be found in thatconfiguration, i.e., fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally found in nature.

The invention provides DNA molecules and their corresponding nucleotidesequences. As used herein, the term “DNA”, “DNA molecule”, “nucleotidemolecule” refers to a DNA molecule of genomic or synthetic origin, i.e.,a polymer of deoxyribonucleotide bases or a polynucleotide molecule,read from the 5′ (upstream) end to the 3′ (downstream) end. As usedherein, the term “DNA sequence”, “nucleotide sequence” or“polynucleotide sequence” refers to the nucleotide sequence of a DNAmolecule. The nomenclature used herein is that required by Title 37 ofthe United States Code of Federal Regulations §1.822 and set forth inthe tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. Byconvention, the nucleotide sequences of the invention provided as SEQ IDNOs: 1-10 and fragments thereof are disclosed with reference to only onestrand of the two complementary nucleotide sequence strands. Byimplication, the complementary sequences (i.e. the sequences of thecomplementary strand), also referred to in the art as the reversecomplementary sequences, are within the scope of the invention and areexpressly intended to be within the scope of the subject matter claimed.

The nucleotide sequence corresponding to the complete nucleotidesequence of the inserted transgenic DNA and substantial segments of thecotton genome DNA flanking either end of the inserted transgenic DNA isprovided herein as SEQ ID NO: 10. A subsection of this is the insertedtransgenic DNA provided as SEQ ID NO: 9. The nucleotide sequence of thecotton genome DNA physically linked by phosphodiester bond linkage toand therefore flanking the 5′ end of the inserted transgenic DNA is setforth as shown in FIG. 1 and provided as SEQ ID NO: 1, 3, 5, and 7. Thenucleotide sequence of the cotton genome DNA physically linked byphosphodiester bond linkage to and therefore flanking the 3′ end of theinserted transgenic DNA is set forth as shown in FIG. 1 and provided asSEQ ID NO: 2, 4, 6, and 8.

The cotton event MON 88701 further comprises two regions, one spanningthe 5′ location and one spanning the 3′ location where the transgenicDNA is inserted into the genomic DNA, referred to herein as the 5′ and3′ junction, respectively. A “junction sequence” or “junction region”refers to the DNA sequence and/or corresponding DNA molecule that spansthe inserted transgenic DNA and the adjacent flanking genomic DNA. Thejunction sequences may be arbitrarily represented by the nucleotidesequences provided as SEQ ID NO: 1 and SEQ ID NO: 2, each representing10 nucleotides of the flanking genomic DNA adjacent to and contiguouswith 10 nucleotides of insert DNA. Alternatively, the junction sequencesmay be arbitrarily represented by the two 60 nucleotide sequencesprovided as SEQ ID NO: 3 and SEQ ID NO: 4, each representing 30nucleotides of the flanking genomic DNA adjacent to and contiguous with30 nucleotides of insert DNA. Alternatively, the junction sequences maybe arbitrarily represented by the two 100 nucleotide sequences providedas SEQ ID NO: 5 and SEQ ID NO: 6, each representing 50 nucleotides ofthe flanking genomic DNA adjacent to and contiguous with 50 nucleotidesof insert DNA. These nucleotides are connected by phosphodiester linkageand in cotton event MON 88701 are present as part of the genome. Theidentification of one or more of SEQ ID NO: 1-10 in a sample derivedfrom a cotton plant, seed, or plant part is determinative that the DNAwas obtained from cotton event MON 88701 and is diagnostic for thepresence in a sample of DNA from cotton event MON 88701. The inventionthus provides a DNA molecule that contains at least one of thenucleotide sequences provided as SEQ ID NO: 1-10. Any segment of DNAderived from transgenic cotton event MON 88701 that is sufficient toinclude at least one of the sequences provided as provided as SEQ ID NO:1-10 is within the scope of the invention. In addition, anypolynucleotide comprising a sequence complementary to any of thesequences described within this paragraph is within the scope of theinvention. FIG. 1 illustrates the physical arrangement of SEQ ID NO: 1-9relative to SEQ ID NO: 10 arranged from 5′ to 3′.

The invention provides exemplary DNA molecules that can be used eitheras primers or probes for diagnosing the presence of DNA derived from acotton plant comprising event MON 88701 in a sample. Such primers orprobes are specific for a target nucleic acid sequence and as such areuseful for the identification of cotton event MON 88701 nucleic acidsequence by the methods of the invention described herein.

A “primer” is typically a highly purified, isolated polynucleotide thatis designed for use in specific annealing or hybridization methods thatinvolve thermal amplification. A pair of primers may be used withtemplate DNA, such as a sample of cotton genomic DNA, in a thermalamplification, such as polymerase chain reaction (PCR), to produce anamplicon, where the amplicon produced from such reaction would have aDNA sequence corresponding to sequence of the template DNA locatedbetween the two sites where the primers hybridized to the template. Asused herein, an “amplicon” is a piece or fragment of DNA that has beensynthesized using amplification techniques. An amplicon of the inventioncomprises at least one of the sequences provided as provided as SEQ IDNO: 1-10. A primer is typically designed to hybridize to a complementarytarget DNA strand to form a hybrid between the primer and the target DNAstrand, and the presence of the primer is a point of recognition by apolymerase to begin extension of the primer (i.e., polymerization ofadditional nucleotides into a lengthening nucleotide molecule) using asa template the target DNA strand. Primer pairs, as used in theinvention, are intended to refer to use of two primers binding oppositestrands of a double stranded nucleotide segment for the purpose ofamplifying linearly the polynucleotide segment between the positionstargeted for binding by the individual members of the primer pair,typically in a thermal amplification reaction or other conventionalnucleic-acid amplification methods. Exemplary DNA molecules useful asprimers are provided as SEQ ID NO: 11-12. The primer pair provided asSEQ ID NO: 11 and SEQ ID NO: 12 are useful as a first DNA molecule and asecond DNA molecule that is different from the first DNA molecule, andboth are each of sufficient length of contiguous nucleotides of SEQ IDNO: 10 to function as DNA primers that, when used together in a thermalamplification reaction with template DNA derived from cotton event MON88701, to produce an amplicon diagnostic for cotton event MON 88701 DNAin a sample.

A “probe” is an isolated nucleic acid that is complementary to a strandof a target nucleic acid. Probes according to the invention include notonly deoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and thedetection of such binding can be useful in diagnosing, discriminating,determining, or confirming the presence of that target DNA sequence in aparticular sample. A probe may be attached to a conventional detectablelabel or reporter molecule, e.g., a radioactive isotope, ligand,chemiluminescent agent, or enzyme. An exemplary DNA molecule useful as aprobe is provided as SEQ ID NO: 13.

Probes and primers according to the invention may have complete sequenceidentity with the target sequence, although primers and probes differingfrom the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of transgenic DNA from cotton event MON 88701 in a sample.Probes and primers are generally at least about 11 nucleotides, at leastabout 18 nucleotides, at least about 24 nucleotides, or at least about30 nucleotides or more in length. Such probes and primers hybridizespecifically to a target DNA sequence under stringent hybridizationconditions. Conventional stringency conditions are described by Sambrooket al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985). As used herein,two nucleic acid molecules are capable of specifically hybridizing toone another if the two molecules are capable of forming ananti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, two molecules exhibit“complete complementarity” if when aligned every nucleotide of the firstmolecule is complementary to every nucleotide of the second molecule.Two molecules are “minimally complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under at least conventional “low-stringency” conditions.Similarly, the molecules are “complementary” if they can hybridize toone another with sufficient stability to permit them to remain annealedto one another under conventional “high-stringency” conditions.Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules normally associated with itin its native or natural state. In one embodiment, the term “isolated”refers to a DNA molecule that is at least partially separated from thenucleic acids which normally flank the DNA molecule in its native ornatural state. Thus, DNA molecules fused to regulatory or codingsequences with which they are not normally associated, for example asthe result of recombinant techniques, are considered isolated herein.Such molecules are considered isolated even when integrated into thechromosome of a host cell or present in a nucleic acid solution withother DNA molecules.

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the invention. For example, PCR (polymerase chain reaction)technology can be used to amplify a particular starting DNA moleculeand/or to produce variants of the original molecule. DNA molecules, orfragment thereof, can also be obtained by other techniques such as bydirectly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying cotton eventMON 88701, selecting plant varieties or hybrids comprising cotton eventMON 88701, detecting the presence of DNA derived from the transgeniccotton event MON 88701 in a sample, and monitoring samples for thepresence and/or absence of cotton event MON 88701 or plant parts derivedfrom cotton plants comprising event MON 88701.

The invention provides cotton plants, progeny, seeds, plant cells, plantparts (such as pollen, ovule, pod, flower tissue, root tissue, stemtissue, and leaf tissue), and commodity products. These plants, progeny,seeds, plant cells, plant parts, and commodity products contain adetectable amount of a polynucleotide of the invention, i.e., such as apolynucleotide having at least one of the sequences provided as SEQ IDNO: 1-10. Plants, progeny, seeds, plant cells, and plant parts of theinvention may also contain one or more additional transgenes. Suchtransgene may be any nucleotide sequence encoding a protein or RNAmolecule conferring a desirable trait including but not limited toincreased insect resistance, increased water use efficiency, increasedyield performance, increased drought resistance, increased seed quality,improved nutritional quality, and/or increased herbicide tolerance, inwhich the desirable trait is measured with respect to a cotton plantlacking such additional transgene.

The invention provides cotton plants, progeny, seeds, plant cells, andplant part such as pollen, ovule, pod, flower, root or stem tissue, andleaves derived from a transgenic cotton plant comprising event MON88701. A representative sample of cotton seed comprising event MON 88701has been deposited according to the Budapest Treaty with the AmericanType Culture Collection (ATCC®). The ATCC repository has assigned thePatent Deposit Designation PTA-11754 to the event MON 88701 comprisingseed.

The invention provides a microorganism comprising a DNA molecule havingat least one sequence selected from SEQ ID NO: 1-10 present in itsgenome. An example of such a microorganism is a transgenic plant cell.Microorganisms, such as a plant cell of the invention, are useful inmany industrial applications, including but not limited to: (i) use asresearch tool for scientific inquiry or industrial research; (ii) use inculture for producing endogenous or recombinant carbohydrate, lipid,nucleic acid, or protein products or small molecules that may be usedfor subsequent scientific research or as industrial products; and (iii)use with modern plant tissue culture techniques to produce transgenicplants or plant tissue cultures that may then be used for agriculturalresearch or production. The production and use of microorganisms such astransgenic plant cells utilizes modern microbiological techniques andhuman intervention to produce a man-made, unique microorganism. In thisprocess, recombinant DNA is inserted into a plant cell's genome tocreate a transgenic plant cell that is separate and unique fromnaturally occurring plant cells. This transgenic plant cell can then becultured much like bacteria and yeast cells using modern microbiologytechniques and may exist in an undifferentiated, unicellular state. Thenew plant cell's genetic composition and phenotype is a technical effectcreated by the integration of the heterologous DNA into the genome ofthe cell. Another aspect of the invention is a method of using amicroorganism of the invention. Methods of using microorganisms of theinvention, such as transgenic plant cells, include (i) methods ofproducing transgenic cells by integrating recombinant DNA into thegenome of the cell and then using this cell to derive additional cellspossessing the same heterologous DNA; (ii) methods of culturing cellsthat contain recombinant DNA using modern microbiology techniques; (iii)methods of producing and purifying endogenous or recombinantcarbohydrate, lipid, nucleic acid, or protein products from culturedcells; and (iv) methods of using modern plant tissue culture techniqueswith transgenic plant cells to produce transgenic plants or transgenicplant tissue cultures.

Plants of the invention may pass along the event DNA, including thetransgene, to progeny. As used herein, “progeny” includes any plant,seed, plant cell, and/or regenerable plant part comprising the event DNAderived from an ancestor plant and/or comprising a DNA molecule havingat least one sequence selected from SEQ ID NO: 1-10. Plants, progeny,and seeds may be homozygous or heterozygous for the transgene. Progenymay be grown from seeds produced by a cotton event MON 88701 containingplant and/or from seeds produced by a plant fertilized with pollen froma cotton event MON 88701 containing plant.

Progeny plants may be self-pollinated (also known as “selfing”) togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene. Selfing of appropriate progeny can produce plants that arehomozygous for both added, exogenous genes.

Alternatively, progeny plants may be outcrossed, e.g., bred with anotherunrelated plant, to produce a varietal or a hybrid seed or plant. Theother unrelated plant may be transgenic or nontransgenic. A varietal orhybrid seed or plant of the invention may thus be derived by crossing afirst parent that lacks the specific and unique DNA of the cotton eventMON 88701 with a second parent comprising cotton event MON 88701,resulting in a hybrid comprising the specific and unique DNA of thecotton event MON 88701. Each parent can be a hybrid or aninbred/varietal, so long as the cross or breeding results in a plant orseed of the invention, i.e., a seed having at least one allelecontaining the DNA of cotton event MON 88701 and/or a DNA moleculehaving at least one sequence selected from SEQ ID NO: 1-10. Twodifferent transgenic plants may thus be crossed to produce hybridoffspring that contain two independently segregating, added, exogenousgenes. For example, the MON 88701 containing dicamba and glufosinatetolerant cotton can be crossed with other transgenic cotton plants toproduce a plant having the characteristics of both transgenic parents.One example of this would be a cross of MON 88701 containing dicamba andglufosinate tolerant cotton with a plant having one or more additionaltraits such as herbicide tolerance (e.g. cotton event MON 1445 or cottonevent MON 88913) and/or insect control (e.g. cotton event MON 15985, MON757, or MON 531), resulting in a progeny plant or seed that is tolerantto dicamba and glufosinate and has at least one or more additionaltraits. Back-crossing to a parental plant and out-crossing with anon-transgenic plant are also contemplated, as is vegetativepropagation. Descriptions of other breeding methods that are commonlyused for different traits and crops can be found in one of severalreferences, e.g., Fehr, in Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

The invention provides a plant part that is derived from cotton plantscomprising event MON 88701. As used herein, a “plant part” refers to anypart of a plant which is comprised of material derived from a cottonplant comprising event MON 88701. Plant parts include but are notlimited to pollen, ovule, pod, flower, root or stem tissue, fibers, andleaves. Plant parts may be viable, nonviable, regenerable, and/ornonregenerable.

The invention provides a commodity product that is derived from cottonplants comprising event MON 88701. As used herein, a “commodity product”refers to any composition or product which is comprised of materialderived from a cotton event MON 88701 containing plant, seed, plantcell, or plant part. Commodity products may be sold to consumers and maybe viable or nonviable. Nonviable commodity products include but are notlimited to nonviable seeds; processed seeds, seed parts, and plantparts; lint; seeds and plant parts processed for feed or food, oil,fiber, paper, biomasses, and fuel products. Viable commodity productsinclude but are not limited to seeds, plants, and plant cells. Thecotton plants comprising event MON 88701 can thus be used to manufactureany commodity product typically acquired from cotton. Any such commodityproduct that is derived from cotton plants comprising event MON 88701may contain at least a detectable amount of the specific and unique DNAcorresponding to cotton event MON 88701, and specifically may contain adetectable amount of a polynucleotide comprising a DNA molecule havingat least one sequence selected from SEQ ID NO: 1-10. Any standard methodof detection for nucleotide molecules may be used, including methods ofdetection disclosed herein. A commodity product is within the scope ofthe invention if there is any detectable amount of a DNA molecule havingat least one sequence selected from SEQ ID NO: 1-10 in the commodityproduct.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the invention are therefore useful for, among other things,growing plants for the purpose of producing seed and/or plant partscomprising cotton event MON 88701 for agricultural purposes, producingprogeny comprising cotton event MON 88701 for plant breeding andresearch purposes, use with microbiological techniques for industrialand research applications, and sale to consumers.

The invention provides methods for controlling weeds and methods forproducing plants using dicamba and glufosinate herbicides and cottonevent MON 88701. A method for controlling weeds in a field is providedand consists of planting cotton event MON 88701 containing varietal orhybrid plants in a field and applying a herbicidally effective dose ofdicamba or glufosinate or dicamba and glufosinate to the field for thepurpose of controlling weeds in the field without injuring the MON 88701containing plants. Such application of dicamba or glufosinate or dicambaand glufosinate herbicides may be pre-emergence, i.e., any time afterMON 88701 containing seed is planted and before MON 88701 containingplants emerge, or post-emergence, i.e., any time after MON 88701containing plants emerge. Another method for controlling weeds in afield is also provided and consists of applying an effective dose ofdicamba or glufosinate or dicamba and glufosinate herbicides to controlweeds in a field and then planting cotton plants comprising event MON88701 in the field. Such application of dicamba or glufosinate ordicamba and glufosinate herbicides would be pre-planting, i.e., beforeMON 88701 containing seed is planted, and could be done any timepre-planting including, but not limited to, about 14 days pre-plantingto about 1 day pre-planting. The invention also provides a method forproducing cotton seed or lint essentially free of weed seeds by plantingseeds of a dicamba and glufosinate tolerant cotton plants comprising MON88701 in a field, applying a post-emergence effective dose of dicamba orglufosinate or dicamba and glufosinate herbicides sufficient to kill theweed to the field, and harvesting seed or lint from the field. Aherbicidally effective dose of dicamba for use in the field shouldconsist of a range from about 0.005 pounds per acre to about 16 poundsper acre of dicamba over a growing season. In one embodiment, a total ofabout 0.5 pounds per acre to about 2 pounds per acre of dicamba isapplied over a growing season. Multiple applications of dicamba may beused over a growing season, for example, two applications (such as apre-planting application and a post-emergence application or apre-emergence application and a post-emergence application) or threeapplications (such as a pre-planting application, a pre-emergenceapplication, and a post-emergence application). A herbicidally effectivedose of glufosinate for use in the field should consist of a range fromabout 0.005 pounds per acre to about 16 pounds per acre of glufosinateover a growing season. In one embodiment, a total of about 0.5 poundsper acre to about 1.59 pounds per acre of glufosinate is applied over agrowing season. Multiple applications of glufosinate may be used over agrowing season, for example, two applications (such as a pre-plantingapplication and a post-emergence application or a pre-emergenceapplication and a post-emergence application) or three applications(such as a pre-planting application, a pre-emergence application, and apost-emergence application or three post-emergence applications).

Methods for producing an herbicide tolerant cotton plant comprising theDNA sequences specific and unique to event MON 88701 of the inventionare provided. Transgenic plants used in these methods may be homozygousor heterozygous for the transgene. Progeny plants produced by thesemethods may be varietal or hybrid plants; may be grown from seedsproduced by a cotton event MON 88701 containing plant and/or from seedsproduced by a plant fertilized with pollen from a cotton event MON 88701containing plant; and may be homozygous or heterozygous for thetransgene. Progeny plants may be subsequently self-pollinated togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene, or alternatively may be outcrossed, e.g., bred with anotherunrelated plant, to produce a varietal or a hybrid seed or plant.

A cotton plant that tolerates application of dicamba and glufosinateherbicides may be produced by sexually crossing an event MON 88701containing plant comprising a DNA molecule having at least one sequenceselected from SEQ ID NO: 1-10 with another cotton plant and therebyproducing seed, which is then grown into progeny plants. These progenyplants may then be treated with dicamba and/or glufosinate herbicides toselect for progeny plants that are tolerant to dicamba and glufosinateherbicides. Alternatively, these progeny plants may be analyzed usingdiagnostic methods to select for progeny plants that contain the eventMON 88701 DNA. The other plant used in the crossing may or may not betolerant to dicamba and glufosinate herbicides and may or may not betransgenic. The progeny plant and/or seed produced may be varietal orhybrid seed. In practicing this method, the step of sexually crossingone plant with another plant, i.e., cross-pollinating, may beaccomplished or facilitated by human intervention, for example: by humanhands collecting the pollen of one plant and contacting this pollen withthe style or stigma of a second plant; by human hands and/or actionsremoving, destroying, or covering the stamen or anthers of a plant(e.g., by detasseling or by application of a chemical gametocide) sothat natural self-pollination is prevented and cross-pollination wouldhave to take place in order for fertilization to occur; by humanplacement of pollinating insects in a position for “directedpollination” (e.g., by placing beehives in orchards or fields or bycaging plants with pollinating insects); by human opening or removing ofparts of the flower to allow for placement or contact of foreign pollenon the style or stigma (e.g., in soy which naturally has flowers thathinder or prevent cross-pollination, making them naturally obligateself-pollinators without human intervention); by selective placement ofplants (e.g., intentionally planting plants in pollinating proximity);and/or by application of chemicals to precipitate flowering or to fosterreceptivity (of the stigma for pollen).

A cotton plant that tolerates application of dicamba and glufosinateherbicides may be produced by selfing an event MON 88701 containingplant comprising a DNA molecule having at least one sequence selectedfrom SEQ ID NO: 1-10 and thereby producing seed, which is then growninto progeny plants. These progeny plants may then be treated withdicamba and glufosinate herbicides to select for progeny plants that aretolerant to dicamba and glufosinate herbicides. Alternatively, theseprogeny plants may be analyzed using diagnostic methods to select forprogeny plants that contain the event MON 88701 DNA. In practicing thismethod, the step of sexually crossing one plant with itself, i.e.,self-pollinating or selfing, may be accomplished or facilitated by humanintervention, for example: by human hands collecting the pollen of theplant and contacting this pollen with the style or stigma of the sameplant and then optionally preventing further fertilization of the plant;by human hands and/or actions removing, destroying, or covering thestamen or anthers of other nearby plants (e.g., by detasseling or byapplication of a chemical gametocide) so that natural cross-pollinationis prevented and self-pollination would have to take place in order forfertilization to occur; by human placement of pollinating insects in aposition for “directed pollination” (e.g., by caging a plant alone withpollinating insects); by human manipulation of the flower or its partsto allow for self-pollination; by selective placement of plants (e.g.,intentionally planting plants beyond pollinating proximity); and/or byapplication of chemicals to precipitate flowering or to fosterreceptivity (of the stigma for pollen).

Progeny cotton plants and seeds encompassed by these methods andproduced by using these methods will be distinct from other cottonplants, for example because the progeny cotton plants and seeds: arerecombinant and as such created by human intervention; are dicamba andglufosinate herbicide tolerant; contain at least one allele thatconsists of the transgene DNA of the invention; and/or contain adetectable amount of a DNA molecule comprising at least one sequenceselected from SEQ ID NO: 1-10. A seed may be selected from an individualprogeny plant, and so long as the seed comprises a DNA molecule havingat least one sequence selected from SEQ ID NO: 1-10, it will be withinthe scope of the invention.

In practicing the invention, two different transgenic plants can becrossed to produce hybrid offspring that contain two independentlysegregating heterologous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of othermethods that are commonly used for different traits and crops can befound in one of several references, e.g., Fehr, in Breeding Methods forCultivar Development, Wilcox J. ed., American Society of Agronomy,Madison Wis. (1987).

The plants and seeds used in the methods disclosed herein may alsocontain one or more additional transgenes. Such transgene may be anynucleotide sequence encoding a protein or RNA molecule conferring adesirable trait including but not limited to increased insectresistance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, improvednutritional quality, and/or increased herbicide tolerance, in which thedesirable trait is measured with respect to a cotton plant lacking suchadditional transgene.

The methods of the invention are therefore useful for, among otherthings, controlling weeds in a field while growing plants for thepurpose of producing seed and/or plant parts comprising cotton event MON88701 for agricultural or research purposes, selecting for progenycomprising cotton event MON 88701 for plant breeding or researchpurposes, and producing progeny plants and seeds comprising cotton eventMON 88701.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the invention may be evaluated for DNA composition, geneexpression, and/or protein expression. Such evaluation may be done byusing any standard method such as PCR, northern blotting, southernanalysis, western blotting, immuno-precipitation, and ELISA or by usingthe methods of detection and/or the detection kits provided herein.

Methods of detecting the presence of DNA derived from a cotton cell,tissue, seed, or plant comprising cotton event MON 88701 in a sample areprovided. One method consists of (i) extracting a DNA sample from atleast one cotton cell, tissue, seed, or plant, (ii) contacting the DNAsample with at least one primer that is capable of producing DNAsequence specific to event MON 88701 DNA under conditions appropriatefor DNA sequencing, (iii) performing a DNA sequencing reaction, and then(iv) confirming that the nucleotide sequence comprises a nucleotidesequence specific for event MON 88701, such as one selected from thegroup consisting of SEQ ID NO: 1-10. Another method consists of (i)extracting a DNA sample from at least one cotton cell, tissue, seed, orplant, (ii) contacting the DNA sample with a primer pair that is capableof producing an amplicon from event MON 88701 DNA under conditionsappropriate for DNA amplification, (iii) performing a DNA amplificationreaction, and then (iv) detecting the amplicon molecule and/orconfirming that the nucleotide sequence of the amplicon comprises anucleotide sequence specific for event MON 88701, such as one selectedfrom the group consisting of SEQ ID NO: 1-10. The amplicon should be onethat is specific for event MON 88701, such as an amplicon that comprisesSEQ ID NO: 1 or SEQ ID NO: 2. The detection of a nucleotide sequencespecific for event MON 88701 in the amplicon is determinative and/ordiagnostic for the presence of the cotton event MON 88701 specific DNAin the sample. An example of a primer pair that is capable of producingan amplicon from event MON 88701 DNA under conditions appropriate forDNA amplification is provided as SEQ ID NO: 11-12. Other primer pairsmay be readily designed by one of skill in the art and would comprise atleast one fragment of SEQ ID NO: 10. Another method of detecting thepresence of DNA derived from a cotton cell, tissue, seed, or plantcomprising cotton event MON 88701 in a sample consists of (i) extractinga DNA sample from at least one cotton cell, tissue, seed, or plant, (ii)contacting the DNA sample with a DNA probe specific for event MON 88701DNA, (iii) allowing the probe and the DNA sample to hybridize understringent hybridization conditions, and then (iv) detectinghybridization between the probe and the target DNA sample. An example ofthe sequence a DNA probe that is specific for event MON 88701 DNA isprovided as SEQ ID NO: 13. Other probes may be readily designed by oneof skill in the art and would comprise at least one fragment of SEQ IDNO: 10. Detection of probe hybridization to the DNA sample is diagnosticfor the presence of cotton event MON 88701 specific DNA in the sample.Absence of hybridization is alternatively diagnostic of the absence ofcotton event MON 88701 specific DNA in the sample.

DNA detection kits are provided that are useful for the identificationof cotton event MON 88701 DNA in a sample and can also be applied tomethods for breeding cotton plants containing the appropriate event DNA.Such kits contain DNA primers and/or probes comprising fragments of SEQID NO: 1-10. One example of such a kit comprises at least one DNAmolecule of sufficient length of contiguous nucleotides of SEQ ID NO: 10to function as a DNA probe useful for detecting the presence and/orabsence of DNA derived from transgenic cotton plants comprising eventMON 88701 in a sample. The DNA derived from transgenic cotton plantscomprising event MON 88701 would comprise a DNA molecule having at leastone sequence selected from SEQ ID NO: 1-10. A DNA molecule sufficientfor use as a DNA probe is provided that is useful for determining,detecting, or diagnosing the presence and/or absence of cotton event MON88701 DNA in a sample is provided as SEQ ID NO: 13. Other probes may bereadily designed by one of skill in the art and should comprise at least15 contiguous nucleotides of SEQ ID NO: 10 and be sufficiently unique tocotton event MON 88701 DNA in order to identify DNA derived from theevent. Another type of kit comprises a primer pair useful for producingan amplicon useful for detecting the presence and/or absence of DNAderived from transgenic cotton event MON 88701 in a sample. Such a kitwould employ a method comprising contacting a target DNA sample with aprimer pair as described herein, then performing a nucleic acidamplification reaction sufficient to produce an amplicon comprising aDNA molecule having at least one sequence selected from SEQ ID NO: 1-10,and then detecting the presence and/or absence of the amplicon. Such amethod may also include sequencing the amplicon or a fragment thereof,which would be determinative of, i.e. diagnostic for, the presence ofthe cotton event MON 88701 specific DNA in the target DNA sample. Otherprimer pairs may be readily designed by one of skill in the art andshould comprise at least 15 contiguous nucleotides of SEQ ID NO: 10 andbe sufficiently unique to cotton event MON 88701 DNA in order toidentify DNA derived from the event.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thermalamplification methods. Many techniques are known in the art fordetecting, quantifying, and/or sequencing the amplicon produced by thesemethods. One exemplary technique useful in practicing this invention isTAQMAN® (PE Applied Biosystems, Foster City, Calif.).

The kits and detection methods of the invention are useful for, amongother things, identifying cotton event MON 88701, selecting plantvarieties or hybrids comprising cotton event MON 88701, detecting thepresence of DNA derived from the transgenic cotton plants comprisingevent MON 88701 in a sample, and monitoring samples for the presenceand/or absence of cotton plants comprising event MON 88701 or plantparts derived from cotton plants comprising event MON 88701.

The sequence of the heterologous DNA insert, junction sequences, orflanking sequences from cotton event MON 88701 can be verified (andcorrected if necessary) by amplifying such sequences from the eventusing primers derived from the sequences provided herein followed bystandard DNA sequencing of the amplicon or of the cloned DNA.

As used herein, the term “comprising” means “including but not limitedto”.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Transformation of Cotton and MON 88701 EventSelection

This example describes the production, analysis, and selection of eventMON 88701. Summarized is the production and analysis of thousands ofindividual plants over multiple years through the rigorous molecular,phenotypic, and field testing required for the ultimate selection of thecommercial event, the MON 88701 event. The transgenic dicamba andglufosinate tolerant cotton event MON 88701 was created throughAgrobacterium-mediated transformation of cotton hypocotyl tissueutilizing a plant transformation vector comprising the expressioncassette illustrated in FIG. 1. Methods for transforming cotton areknown in the art. To produce the MON 88701 event, Coker 130 cottonmaterial (Asgrow, St Louis, Mo.) was used for plant transformation.Cotton cells were transformed and regenerated into intact cotton plants.Rooted plants with normal phenotypic characteristics were selected andtransferred to soil for growth and further assessment.

R0 plants were transferred to soil and subjected to herbicide screeningat 1× or 2× field rates (i.e., 0.5 or 1.0 lb/acre active ingredient) ofboth dicamba and glufosinate. Subsequently, R0 plants containing only asingle T-DNA expression cassette were identified and selected. The T-DNAexpression cassette contains the Peanut chlorotic streak virus (PClSV)promoter with a duplicated enhancer region (P-PClSV.FLt-enh); operablylinked to a DNA leader derived from RNA transcript of Tobacco etch virus(L-TEV); operably linked to a DNA molecule encoding an N-terminalchloroplast transit peptide from chloroplast transit peptide from5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Arabidopsisthaliana (TS-CTP2); operably linked to a DNA molecule encoding dicambamono-oxygenase (DMO) from Stenotrophomonas maltophilia; operably linkedto a 3′ UTR DNA molecule derived from the fiber protein E6 gene ofsea-island cotton (T-Gb.E6-3b); operably linked to the promoter of theenhanced 35S RNA transcript (with a duplicated enhancer region) fromCauliflower mosaic virus (P-CaMV.35S-enh); operably linked to a DNAleader derived from the 5 untranslated region of the heat shock protein70 (HSP70) gene from Petunia x hybrida (L-Ph.DnaK); operably linked tothe Bialaphos Resistance gene (bar) gene from Streptomyces hygroscopicusencoding a phosphinothricin acetyl transferase (PAT) (CR-Sh.bar);operably linked to a 3′ untranslated region from the nopaline synthase(NOS) gene (which directs polyadenylation of mRNA) from Agrobacteriumtumefaciens (T-AGRtu.nos).

The MON 88701 event was selected from among approximately 151 individualtransgenic events based on its superior combination of phenotypiccharacteristics (including commercial level tolerance to both dicambaand glufosinate herbicides), molecular characteristics (determined usinga comprehensive molecular profile analysis), and haplotype association.Selection of the MON 88701 event from among the original 151 transgenicevents was a multi-year process requiring data analysis at each step.Two waves of transformation events were processed to allow for a largernumber of events from which to choose the final commercial event. Table1 provides a summary of each type of analysis with the number of eventsselected and advanced for each step of event screening. The Puerto Ricofield trial efficacy screen for Wave 2 preceded the first U.S. fieldtrial.

TABLE 1 Event Screening Analysis Step Wave 1 Wave 2 R0—TransformationQuality 48 103 Analysis R0—Herbicide Efficacy Analysis 26 57R1—Agronomic Seed Production 9 26 R1—Herbicide Efficacy Analysis 9 23First U.S. Field Trial 8 10 Preliminary Molecular Analysis 4 19 PuertoRico Field Trial Efficacy 3 17 Screen Second U.S. Field Trial 2 n/dDetailed Molecular Analysis 1 6

The original 151 transgenic R0 events were analyzed using a combinationof analytical techniques (Transformation Quality Analysis includingTaqMan and/or PCR analysis) and R0 Herbicide Efficacy Analysis(herbicide spray). Selected R0 event containing plants were thenself-fertilized to produce R1 seed, and 32 of the events were thenadvanced for R1 Herbicide Efficacy Analysis.

Detailed molecular analysis was completed on events passing agronomicand efficacy field trials. Northern blots were used to confirm that thedmo and bar messenger RNA transcripts were present in samples preparedfrom seed of the tested events. Western blot analysis was done toconfirm that a single DMO and PAT protein band was present in samplesprepared from leaf tissue of the tested events. N-terminal sequencing ofthe isolated, purified PAT protein showed the expected amino acidsequence at the N-terminus of the PAT protein. N-terminal sequencing ofthe isolated, purified DMO protein showed 9 amino acids from the CTP(TS-CTP2) present at the N-terminus of the DMO protein.

Agronomic field testing consisted of planting paired positive isolineand negative isoline events (containing the transgene cassette andlacking the transgene cassette, respectively) side-by-side with two rowsof wild type (non-transgenic) Coker 130 per plot. The plots weremaintained weed and insect free using conventional control programs forthe duration of the trial. Data collected included planting date, standcounts, plant height, node number, gross agronomic/phenotypicdifferences, boll samples, harvest date, and yield. Stand counts foreach plot were taken at 10 days after planting (DAP) and again at 20DAP. Cotyledons that had completely cleared the soil were considered“emerged”. Any differences noted in plant density, i.e. skips, poorgermination, loss due to crusting, washouts, etc., were noted. Finalstands had 10 plants per meter on average. Plant height measurementswere taken at first flower plus two weeks (FF2). Average measurements ofplant height (cm) of five plants per plot were recorded. On the sameplants used for plant height measurements, the number of nodes werecounted (cotyledon is node 0). Any gross agronomic differences wererecorded, such as leaf morphology, branching habit, leaf color, time toflowering, fruiting pattern, etc. Boll samples were randomly harvestedfrom 50 first position bolls (seedcotton only) from the middle of theplants (notes 8-12). The boll samples were used to calculate lintfraction and fiber quality. For yield determinations, plots weredefoliated and a boll opener (with ethephon, i.e. PREP) was used priorto harvest. Plots were harvested and recorded as pounds per plot ofseedcotton and expressed as pounds per acre of seedcotton. There was nosignificant yield difference between positive isoline events, the pairednegative isoline events, and wild-type Coker 130 cotton for two years ofagronomic field trials (FIG. 2).

In year 1, efficacy testing was conducted with eight events compared tonon-transgenic, wild-type Coker 130. For this testing, paired plots weresprayed with dicamba (Clarity®, BASF, Research Triangle Park, N.C.) andglufosinate (Ignite®, Bayer CropScience, Research Triangle Park, N.C.)or left unsprayed. The plots were maintained weed- and insect-free usingconventional control programs for the duration of the trial. Allherbicide treatments are represented as pounds of active ingredient peracre (lb/A). For plots that were sprayed, the following applicationswere conducted: dicamba was applied preemergence at 1 lb/A followed byglufosinate applied 1.09 lb/A at the 2 node stage, followed by dicambaapplied at 1 lb/A at the 5 node stage, followed by glufosinate applied1.09 lb/A at the 8 node stage, followed by dicamba applied at 1 lb/A atthe 12 node stage. Neither dicamba nor glufosinate were applied withsurfactants, fertilizer additive, or other adjuvant. Injury ratings weretaken at the following stages, which were immediately prior to the nextassigned herbicide application: at the 2-node cotton stage, ratingactivity was taken for percent epinasty (dicamba damage) and percenttotal injury, with focus on epinasty; at the 5-node stage, ratingactivity was taken for percent epinasty, percent burn or necrosis(glufosinate injury) and percent total injury, with focus on necrosis;at the 8-node stage, rating activity was taken for percent epinasty,percent burn or necrosis, and percent total injury, with focus onepinasty; at the 12-node stage, rating activity was taken for percentepinasty, percent burn or necrosis and percent total injury, with focuson necrosis; at mid-bloom stage, rating activity was taken for percentepinasty, percent burn or necrosis and percent total injury, with focuson epinasty; and for late bloom stage, rating activity was taken forpercent epinasty, percent burn or necrosis and percent total injury.Additional agronomic data were collected in these efficacy field trialsincluding: planting date, stand counts (taken at 7 and 14 DAP), grossagronomic/phenotypic differences, harvest date, and yield. Injuryratings used standard weed science percent scales where 0% equals nocrop injury and 100% equals complete crop death. Ratings made fordicamba injury are referred to as epinasty and appears as twisting,growth malformation. Ratings made for glufosinate injury are referred toas burn or necrosis and appears as chlorosis and or necrosis. For yielddeterminations, plots were defoliated and a boll opener (with ethephon,i.e. PREP) was used prior to harvest. Plots were harvested and yieldrecorded as lbs/plot of seed cotton and expressed as lb/acre of seedcotton. Agronomic/phenotypic differences were notedin fruiting profileas normal or as unusual. There was no significant difference in yield ofseed cotton between paired sprayed and unsprayed events compared tounsprayed wild-type Coker 130 (FIG. 3). The wild-type Coker 130 did notsurvive the spray regimen (FIG. 7).

In year 2, efficacy testing was conducted with twelve events compared tonon-transgenic, wild-type Coker 130. For this testing, paired plots wereeither sprayed or not sprayed. The plots were maintained weed- andinsect-free using conventional control programs for the duration of thetrial. For the plots that were sprayed, the following treatmentapplications were conducted (1) 0.5 lb/A dicamba applied PRE, EarlyPOST, Mid POST and Late POST; (2) 1 lb/A dicamba applied PRE, EarlyPOST, Mid POST and Late POST; (3) 2 lb/A dicamba applied PRE, EarlyPOST, Mid POST and Late POST; (4) 0.5 lb/A glufosinate applied EarlyPOST, Mid POST and Late POST: (5) 1 lb/A glufosinate applied Early POST,Mid POST and Late POST: 6) 2 lb/A glufosinate applied Early POST, MidPOST and Late POST; and 7) Control (unsprayed). Preemergence (PRE) isdefined as immediately after cotton planting (within 24 hours); EarlyPOST as 2-node cotton; Mid POST as 8-node cotton; and Late POST as 14node cotton. For these efficacy field trials, there were three plotscomposed entirely of wild-type cotton (Coker 130). At eachpost-emergence timing (Early, Mid, Late), one-half of a plot was sprayedwith the 1× rate (0.5 lb/A) of dicamba and one-half of the same plot wassprayed with the 1× rate (0.5 lb/A) of glufosinate. Each of the threeplots received dicamba and glufosinate spray at one of thepost-emergence timing spray applications (Early, Mid, Late).

The spray protocol consisted of the herbicides applied at 10 gallons peracre water volume using the following sprayer set up (which is withinthe standards used for most weed science research). XR TeeJet® extendedrange flat spray tips with an 80-degree spray angle were used (TeeJetTechnologies, Wheaton, Ill.). There were 2 nozzles per row (nozzlesspaced at 15″ for 30″ rows, 19″ for 38″ rows and 20″ for 40″ rows).Nozzle size depended on sprayer speed and nozzle spacing; however,nozzle size was selected to result in a spray pressure of 25 to 35 PSI.Boom height was adjusted to allow for 50% spray overlap (which occurs atroughly 30″ above the top of the crop).

At each rating time, the following observations were recorded: 1)percent necrosis or burn on the most affected leaves; 2) percentnecrosis or burn on a whole-plant basis; 3) epinasty; and 4) any otherrelevant injury that was observed. Ratings were taken four days aftereach application and immediately prior to subsequent applications. Therating schedule time and objective were as follows: 1) 4-7 days afteremergence and again immediately before 2-node spray to check forpreemergence effects of dicamba spray; 2) 4 days after the 2-node sprayand again immediately before the 8-node spray to determine the rating ofthe 2-node spray; 3); 4 days after the 8-node spray and againimmediately before the 14-node spray to determine the rating of the8-node spray 4); 4 days after the 14-node spray and again 10 days afterthe 14-node spray to determine the rating of the 14-node spray; and 5)at cutout to determine the late season rating of the net effect of allsprays. Injury ratings used standard weed science percent scales where0% equals no crop injury and 100% equals complete crop death. Ratingsmade for dicamba injury are referred to as epinasty and appears astwisting, growth malformation. Ratings made for glufosinate injury arereferred to as burn or necrosis and appears as chlorosis and ornecrosis. For yield determinations, plots were defoliated and a bollopener (with ethephon, i.e. PREP) was used prior to harvest. Plots wereharvested and recorded as lbs/plot of seedcotton and expressed aslb/acre of seedcotton. Wild-type Coker 130 plants did not surviveherbicide spray and therefore no yield was recorded from Coker 130plots. As seen in FIG. 4.A, yield was nearly equivalent for plantscomprising the MON 88701 event with increasing application rates ofdicamba herbicide. As seen in FIG. 4.B, yield was nearly equivalent forplants comprising the MON 88701 event with increasing application ratesof glufosinate herbicide. For injury ratings, FIG. 5.A shows increasedpercentage of injury to plants comprising the MON 88701 event withincreasing application rates of dicamba. FIG. 5.B shows increasedpercentage of injury to plants comprising the MON 88701 event withincreasing application rates of glufosinate.

In year 3, efficacy field testing of event MON 88701 was conducted tostudy the rate and timing of application of dicamba and/or glufosinate.The plots were maintained weed- and insect-free using conventionalcontrol programs for the duration of the trial. The treatment andapplication timing are detailed in Table 2. Neither dicamba norglufosinate were applied with surfactants, fertilizer additive or otheradjuvant. The sprayer protocol was as described for year 2 efficacyfield testing. Stand rating, visual injury rating (percent epinasty,percent necrosis), plant height and node number, 50-boll sampling, andyield determinations were as described for agronomic and efficacy fieldtesting above.

TABLE 2 Rate Application timing Treat- (lb 10 to 12 15 to 18 ment ai/A)1 to 3 Node 5 to 7 node node node 1 0.5 Dicamba Glufosinate DicambaGlufosinate 2 1 Dicamba Glufosinate Dicamba Glufosinate 3 0.5Glufosinate Dicamba Glufosinate Dicamba 4 1 Glufosinate DicambaGlufosinate Dicamba 5 0.25* Tank Mix Tank Mix Tank Mix Tank Mix 6 0.5*Tank Mix Tank Mix Tank Mix Tank Mix 7 1* Tank Mix Tank Mix Tank Mix TankMix 8 0.5 Dicamba Dicamba Dicamba Dicamba 9 1 Dicamba Dicamba DicambaDicamba 10 0.5 Glufosinate Glufosinate Glufosinate Glufosinate 11 1Glufosinate Glufosinate Glufosinate Glufosinate 12 — Control — — — *Withtank mixes, the rate is what was used for the individual herbicides:0.25 = 0.25 lb/A dicamba + 0.25 lb/A glufosinate

There was a slight yield drag when dicamba and glufosinate were tankmixed and applied at four times in season at 1 lb/acre each (FIG. 6).For all other treatment regimens, there was no yield loss to plantscomprising the MON 88701 event when sprayed with dicamba, glufosinate,or dicamba and glufosinate (FIG. 6).

Example 2 Characterization of MON 88701 DNA Sequences

This example describes the molecular characterization of the MON 88701event. The DNA inserted into the genome of cotton plants comprising MON88701 and the genomic sequence flanking this insertion werecharacterized by detailed molecular analyses. These analyses included:the insert sequence, the insert number (number of integration siteswithin the cotton genome), the copy number (number of copies oftransgene DNA within one locus), the integrity of the inserted genecassette, the flanking sequences, and the association of the insertionwith haplotype regions of the cotton genome.

Molecular DNA probes were used that included the intact coding regionand its respective regulatory elements, the promoters, introns, andpolyadenylation sequences of the plant expression cassettes. Theanalysis showed that MON 88701 contains a single transgene DNA insertionwith one copy of the expression cassette. Inverse PCR and DNA sequenceanalyses were performed to determine the 5′ and 3′ insert-to-plantgenome junctions, confirm the organization of the elements within theinsert (FIG. 1), and determine the complete DNA sequence of the insertin cotton plants comprising event MON 88701 (provided herein as SEQ IDNO: 9). A cotton plant that comprises in its genome the linked transgenegenetic elements shown in FIG. 1 and is resistant to dicamba andglufosinate herbicides is an aspect of the invention.

Sequences flanking the transgene DNA insertion in MON 88701 weredetermined using inverse PCR as described in Ochman et al., 1990 (PCRProtocols: A guide to Methods and Applications, Academic Press, Inc.)and/or genome walker techniques. Plant genomic DNA was isolated fromboth Coker 130 and the transgenic cotton lines from tissue grown understandard greenhouse conditions. Approximately 0.2 gram of young leaftissue was lyophilized and powdered via the addition of several smallsteel beads followed by vigorous agitation. Tissue was washed twice with5 mL of 100% methanol followed by one wash with 5 mL of 50% methanol toremove interfering phenolic compounds. DNA was extracted by the additionof 7 mL of CTAB extraction buffer (100 mM Tris pH 8.0, 1.4 M NaCl, 20 mMEDTA, 2% w/v hexadecyltrimethylammonium bromide, 1% w/vpolyvinylpyrrolidone, 1% w/v poly(vinylpolypyrrolidone), 0.07%beta-mercaptoethanol, 1 mM DTT). Samples were incubated at 65° C. for 45min to fully lyse cells then, spun for 5 min at 3000 g to removeinsoluble debris. The supernatant was extracted with 7 mL of chloroformisoamyl alcohol 24:1, spun at 3000 g for 5 min to separate phases, andDNA was extracted from the aqueous phase in a fresh tube by the additionof 6 mL of isopropanol. DNA pellets were washed with 6 mL of 70% ethanolto remove any salts, and pellets were allowed to air dry. The DNApellets were resuspended in 10 mM Tris pH 8.0 containing 0.5 ug/mLDNase-free RNase and incubated at 37° C. 1 hour. Following treatment,DNA was quantified using Quant-iT™ PicoGreen® (Molecular Probes, Inc.Eugene, Oreg.). This method can be modified by one skilled in the art toextract DNA from any tissue of cotton. An aliquot of DNA was digestedwith restriction endonucleases selected based upon restriction analysisof the transgene DNA. After self-ligation of restriction fragments, PCRwas performed using primers designed from the transgene DNA sequencethat would amplify sequences extending away from the 5′ and 3′ ends ofthe transgene DNA. PCR products were separated by agarose gelelectrophoresis and purified using a QIAGEN gel purification kit(Qiagen, Valencia, Calif.). The subsequent DNA products were sequenceddirectly using standard DNA sequencing protocols. The 5′ flankingsequence which extends into the right border sequence of the expressioncassette transgene DNA is presented as SEQ ID NO: 7 ([C], see FIG. 1).The 3′ flanking sequence which extends into the left border sequence ofthe expression cassette transgene DNA is presented as SEQ ID NO: 8 ([D],see FIG. 1). The portion of the expression cassette DNA that was fullyintegrated into the Coker genomic DNA is presented as SEQ ID NO: 9 ([E],see FIG. 1).

Isolated DNA molecule sequences were compared to the transgene DNAsequence to identify the flanking sequence and the co-isolated transgeneDNA fragment. Confirmation of the presence of the expression cassettewas achieved by PCR with primers designed based upon the deducedflanking sequence data and the known transgene DNA sequence. Thewild-type sequence corresponding to the same region in which thetransgene DNA was integrated in the transformed line was isolated usingprimers designed from the flanking sequences in MON 88701. The PCRreactions were performed using the Phusion® High-Fidelity PCR Master Mixwith HF Buffer (New England Biolabs, Ipswich, Mass.). The flanking DNAsequences in MON 88701 and the Coker wild-type sequence were analyzedagainst multiple nucleotide and protein databases. This information wasused to examine the relationship of the transgene to the plant genomeand to look for the insertion site integrity. The flanking sequence andwild-type sequences were used to design primers for TAQMAN® endpointassays used to identify the events. Zygosity assays were developed usingthis information.

The dicamba and glufosinate tolerance transgene cassette was mapped incotton plants comprising event MON 88701 on chromosome A08 at the mapposition of 19.3 cM and bordered by NG0207927 at the map position of18.6 cM on left and by NG0207529 at the map position of 20.0 cM onright.

Example 3 Event Specific Endpoint TAQMAN® Assays

This example describes an event specific endpoint TAQMAN® thermalamplification method developed to identify event MON 88701 in a sample.Examples of conditions useful with the event MON 88701 Specific EndpointTAQMAN® method are as follows: Step 1: 18 megohm water adjusted forfinal volume of 10 μl. Step 2: 5.0 μl of 2× Universal Master Mix (dNTPs,enzyme, buffer) to a 1× final concentration. Step 3: 0.5 μl EventPrimer-1 (SQ21654) and Event Primer-2 (SQ23205). Mix (resuspended in 18megohm water to a concentration of 20 uM for each primer) to 1.0 μMfinal concentration (for example in a microcentrifuge tube, thefollowing should be added to achieve 500 μl at a final concentration of20 uM: 100 μl of Primer SQ21654 at a concentration of 100 μM; 100 μl ofPrimer SQ23205 at a concentration of 100 μM; 300 μl of 18 megohm water).Step 4: 0.2 μl Event 6-FAM™ MGB Probe PB10280 (resuspended in 18 megohmwater to a concentration of 10 μM to 0.2 μM final concentration. Step 5:0.5 μl Internal Control Primer-1 and Internal Control Primer-2 Mix(resuspended in 18 megohm water to a concentration of 20 μM for eachprimer) to 1.0 μM final concentration. Step 6: 0.2 μl Internal ControlVIC™ Probe to 0.2 μM final concentration (resuspended in 18 megohm waterto a concentration of 10 μM) Step 7: 3.0 μl Extracted DNA (template) foreach sample with one each of the following comprising 1. Leaf Samples tobe analyzed; 2. Negative control (non-transgenic DNA); 3. Negative watercontrol (no template); 4. Positive control MON 88701 DNA. Step 8:Thermocycler Conditions as follows: One Cycle at 50° C. for 2 minutes;One Cycle at 95° C. for 10 minutes; Ten Cycles of 95° C. for 15 secondsthen 64° C. for 1 minute with −1° C./cycle; Thirty Cycles of 95° C. for15 seconds then 54° C. 1 minute, optional additional 10 to 20 cycles(95° C. for 15 seconds then 64° C. for 1 minute (−1° C./cycle) mayprovide more distinct population separation during EndPoint TaqMan®analysis; final cycle of 10° C.

The DNA primers used in the endpoint assay are primers SQ21654 (SEQ IDNO: 11), SQ23205 (SEQ ID NO: 12), and 6-FAM™ labeled probe PB10280 (SEQID NO: 13). 6-FAM™ is a fluorescent dye product of Applied Biosystems(Foster City, Calif.) attached to the DNA probe. For TAQMAN® MGB™probes, the 5′ exonuclease activity of Taq DNA polymerase cleaves theprobe from the 5′-end, between the fluorophore and quencher. Whenhybridized to the target DNA strand, quencher and fluorophore areseparated enough to produce a fluorescent signal, thus releasingfluorescence. SQ21654 (SEQ ID NO: 11) and SQ23205 (SEQ ID NO: 12) whenused with these reaction methods with PB10280 (SEQ ID NO: 13) produce aDNA amplicon that is diagnostic for event MON 88701 DNA. The controlsfor this analysis should include a positive control from cottoncontaining event MON 88701 DNA, a negative control from non-transgeniccotton, and a negative control that contains no template DNA.Additionally, a control for the PCR reaction includes Internal ControlPrimers and an Internal Control Probe, specific to a single copy gene inthe Gossypium genome. One of skill in the art will know how to designprimers specific to a single copy gene in the Gossypium genome. Theseassays are optimized for use with either an Applied Biosystems GeneAmp®PCR System 9700 (run at maximum speed) or MJ Research DNA Engine PTC-225thermal cycler. Other methods and apparatus known to those skilled inthe art that produce amplicons that identify the event MON 88701 DNA iswithin the skill of the art.

R0 plants demonstrating the presence of the expression cassette wereallowed to develop into fully mature plants and seed was harvested fromthese. Probes based on the sequence of the expression cassette were usedto determine copy number of the transgenic expression cassette in the R1plants using a combination of Southern analysis and endpoint TAQMAN®.

A zygosity assay is useful for determining if a plant comprising anevent is homozygous for the event DNA; that is comprising the exogenousDNA in the same location on each chromosome of a chromosomal pair; orheterozygous for an event DNA, that is comprising the exogenous DNA ononly one chromosome of a chromosomal pair; or is null for the event DNA,that is wildtype. The endpoint TAQMAN® thermal amplification method wasalso used to develop zygosity assays for event MON 88701. This exampledescribes an event-specific endpoint TAQMAN® thermal amplificationmethod developed to determine the zygosity of event MON 88701 in asample. For this assay, a three primer assay was employed wherein primerSQ21654 hybridizes and extends specifically from the 3′ junction of theinserted exogenous DNA and genomic DNA, primer SQ23205 hybridizes andextends specifically from the DNA flanking the 3′ side of the insertedexogenous DNA, and primer SQ23901 hybridizes and extends specificallyfrom genomic DNA into which was integrated the inserted exogenous DNA.The three primers are diagnostic for the event. In this example, primerSQ21654 and primer SQ23205 and the 6-FAM™-labeled oligonucleotide probePB10280 are diagnostic when there is a copy of the inserted exogenousDNA. In this example, SQ23205 and primer SQ23901 and the VIC™-labeledoligonucleotide probe PB10631 are diagnostic when there is no copy ofthe inserted exogenous DNA present in the genomic DNA, i.e. wild-type.When the three primers and two probes are mixed together in a PCRreaction with DNA extracted from a plant homozygous for event MON 88701,there is a fluorescent signal only from 6-FAM™-labeled oligonucleotideprobe PB10280 which is indicative of and diagnostic for a planthomozygous for event MON 88701. When the three primers and two probesare mixed together in a PCR reaction with DNA extracted from a plantheterozygous for event MON 88701, there is a fluorescent signal fromboth the 6-FAM™-labeled oligonucleotide probe PB10280 and theVIC™-labeled oligonucleotide probe PB10631 which is indicative of anddiagnostic for a plant heterozygous for event MON 88701. When the threeprimers and two probes are mixed together in a PCR reaction with DNAextracted from a plant which is null for event MON 88701 (i.e.wildtype), there is a fluorescent signal from only the VIC™-labeledoligonucleotide probe PB10631 which is indicative of and diagnostic fora plant null for event MON 88701, i.e. wild-type. Examples of conditionsuseful with this method are as follows. Step 1: 18 megohm water adjustedfor final volume of 10 μl. Step 2: 5.0 μl of 2× Universal Master Mix(Applied Biosystems cat #4304437; dNTPs, enzyme, buffer) to a 1× finalconcentration. Step 3: 0.5 μl of Zygosity Primers SQ21654; SQ23205 andSQ23901 (resuspended in 18 megohm water to a concentration of 20 μM foreach primer) to a final concentration of 1.0 μM. Step 4: 0.2 μl 6-FAM™Probe PB10280 (resuspended in 18 megohm water to a concentration of 10μM) to 0.2 μM final concentration. Step 5: 0.2 μl VIC™ Probe PB10631(resuspended in 18 megohm water to a concentration of 10 μM) to 0.2 μMfinal concentration. Step 6: 3.0 μl Extracted DNA (template) for eachsample with one each of the following comprising 1. Leaf Samples to beanalyzed (4-5 ng/ul of genomic DNA diluted in water); 2. Negativecontrol (non-transgenic cotton DNA; 5 ng/ul diluted in water); 3.Negative water control (no template; solution in which DNA wasresuspended); 4. Positive control MON 88701 genomic DNA from knownheterozygous sample (5 ng/ul diluted in water); 5. 4. Positive controlMON 88701 genomic DNA from known homozygous sample (5 ng/ul diluted inwater). Step 7: Gently mix. Step 8: Thermocycler Conditions when usingApplied Biosystems GeneAmp® PCR System 9700 (run at maximum speed) or MJResearch DNA Engine PTC-225 thermal cycler are as follows: One Cycle at50° C. for 2 minutes; one cycle at 95° C. for 10 minutes; Ten Cycles of(95° C. for 15 seconds then 64° C. for 1 minute (−1° C./cycle); ThirtyCycles of (95° C. for 15 seconds then 54° C. for 1 minute); Optionaladditional 10 to 20 cycles (95° C. for 15 seconds then 64° C. for 1minute (−1° C./cycle) may provide more distinct population separationduring EndPoint TaqMan® analysis; One cycle at 10° C. hold.

Example 4 Identification of Event MON 88701 in any Cotton BreedingActivity

This example describes how one may identify the MON 88701 event withinprogeny of any cotton breeding activity. DNA event primer pairs are usedto produce an amplicon diagnostic for cotton event MON 88701. Anamplicon diagnostic for MON 88701 comprises at least one of thesequences provided as SEQ ID NO: 1-10. Event primer pairs that willproduce a diagnostic amplicon for MON 88701 include primer pairs basedupon the flanking sequences and the inserted expression cassette. Forexample, to produce a diagnostic amplicon in which SEQ ID NO: 1 isfound, one would design a forward primer based upon the genomic flankingsequence portion of SEQ ID NO: 7 and a reverse primer based upon theinserted expression cassette DNA sequence with the primers of sufficientlength of contiguous nucleotides to specifically hybridize to the eventDNA. To produce a diagnostic amplicon in which SEQ ID NO: 2 is found,one would design a forward primer based upon the inserted expressioncassette DNA sequence and a reverse primer based upon the genomicflanking sequence portion of SEQ ID NO: 8 with the primers of sufficientlength of contiguous nucleotides to specifically hybridize to the eventDNA. For practical purposes, one should design primers which produceamplicons of a limited size range, for example, between 100 to 1000bases. Smaller (shorter polynucleotide length) sized amplicons ingeneral are more reliably produced in PCR reactions, allow for shortercycle times, and can be easily separated and visualized on agarose gelsor adapted for use in endpoint TAQMAN®-like assays. Smaller ampliconscan be produced and detected by methods known in the art of DNA amplicondetection. In addition, amplicons produced using the primer pairs can becloned into vectors, propagated, isolated, and sequenced or can besequenced directly with methods well established in the art. Any primerpair that is useful in a DNA amplification method to produce an amplicondiagnostic for MON 88701 or plants comprising MONS 88701 or progenythereof is an aspect of the invention. An example of the amplificationconditions for this analysis is illustrated in Example 3.

An analysis for event MON 88701 containing plant tissue sample shouldinclude a positive tissue control from a plant comprising event MON88701, a negative control from a cotton plant that does not containevent MON 88701 (for example, but not limited to Coker), and a negativecontrol that contains no cotton genomic DNA. A primer pair that willamplify an endogenous cotton DNA molecule will serve as an internalcontrol for the DNA amplification conditions. Additional primersequences can be selected from SEQ ID NO: 10 by those skilled in the artof DNA amplification methods, and conditions selected for the productionof an amplicon by the methods shown in Example 3 may differ, but resultin an amplicon diagnostic for event MON 88701 DNA. DNA detection kitscontain at least one DNA primer of sufficient length of contiguousnucleotides derived from SEQ ID NO: 10, that when used in a DNAamplification method produces a diagnostic amplicon for MON 88701 is anaspect of the invention.

A deposit of a representative sample of cotton seed comprising event MON88701 has been made according to the Budapest Treaty with the AmericanType Culture Collection (ATCC) having an address at 10801 UniversityBoulevard, Manassas, Va. USA, Zip Code 20110. The ATCC Patent DepositDesignation (accession number) for seeds comprising event MON 88701 isPTA-11754 and the date of deposit was Mar. 17, 2011. Access to thedeposits will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced as necessary during that period.

Having illustrated and described the principles of the invention, itshould be apparent to persons skilled in the art that the invention canbe modified in arrangement and detail without departing from suchprinciples. We claim all modifications that are within the spirit andscope of the appended claims.

1-29. (canceled)
 30. A method of determining the zygosity of a cottonplant or seed comprising event MON 88701 comprising: a) contacting asample comprising cotton DNA with a primer set and a probe set that i)when used together in a nucleic acid amplification reaction with eventMON 88701 DNA, produces a first amplicon that is diagnostic for eventMON 88701; and ii) when used together in a nucleic acid amplificationreaction with cotton genomic DNA other than event MON 88701 DNA,produces a second amplicon that is diagnostic native cotton genomic DNAother than event MON 88701; b) performing a nucleic acid amplificationreaction with said sample, primer set, and probe set; c) detecting insaid nucleic acid amplification reaction a first fluorescent signal thatis diagnostic for event MON 88701 and a second fluorescent signaldifferent from said first fluorescent signal and diagnostic for nativecotton genomic DNA corresponding to the location of insertion of theevent MON 88701 transgene; and d) analyzing the presence or absence ofsaid first fluorescent signal and said second fluorescent signal in saidnucleic acid amplification reaction, wherein the presence of bothfluorescent signals indicates said sample is heterozygous for event MON88701 and the presence of only said first fluorescent signal indicatessaid sample is homozygous for event MON
 88701. 31. The method of claim30, wherein the primer set comprises SEQ ID NO: 11, SEQ ID NO: 12 andSEQ ID NO:
 14. 32. The method of claim 30, wherein the probe setcomprises SEQ ID NO: 13 and SEQ ID NO:
 15. 33. (canceled)